The present invention relates to a carrier-bound immunosorbent comprising a carrier body carrying an immobilised immuno reactant.
Immobilised antibodies cross-linked by covalent bonding, e.g. with a cross-linking agent such as glutaraldehyde, to a carbohydrate type of carrier, e.g. cellulose or agarose are known (R. Kowal et alia, anal. Biochem. 102, 72-76 (1980)). The carrying capacity of these carbohydrates is comparatively low and the products are not very stable.
T. T. Ngo (Int. J. Biochem Vol. 11, pages 459-465) describes a variety of immobilised enzyme systems--but not in the context of immuno reagents--in which the enzymes are immobilised
(a) by adsorption (such enzymes becoming easily desorbed); PA0 (b) by covalent attachment of their non-essential amino acid residues to "chemically activated supports" such as cellulose, glass or synthetic polymers via azide, isocyanate, carbodiimide and other derivatives of the supports, PA0 (c) polycondensation by cross-linking of the enzymes themselves, using bifunctional cross-linking agents such as diisothyocyanate, bisimidates, alkylating agents and dialdehydes to form aggregates in which, however, a major part of the desired characteristics of the enzymes is lost. PA0 Immunogen: An immunogen is a foreign substance e.g. protein or particle, e.g. virus or bacterium, which can by itself. induce an immune response which usually results in the appearance of antibodies (Ab) e.g. in the circulation of an animal the immuno system of which is exposed to the immunoger, e.g. by injection. PA0 Antigen: An antigen (Ag) is a substance or particle which can bind to its specific antibody to form an Ab-Ag complex, called an immune complex, but which will not necessarily induce an immune response by itself. PA0 Hapten: A hapten is a substance, generally of low molecular weight, incapable of eliciting antibody formation by itself, but able to react with an antibody and to become immunogenic when coupled to a carrier. PA0 Immuno-reactant: a substance capable of being one of the reaction partners in an antibody/antigen (or hapten) complex formation. PA0 (a) if the immuno reactant is not available in sufficiently large quantities to provide all the film-forming material needed; and PA0 (b) if the immuno reactant itself taken alone does not form a satisfactory film. In that case the immuno reactant may be attached covalently onto a film-forming material, e.g. with one or more bifunctional reagents having at least one functional group reactive to the additional film-forming substance and at least one functional group reactive to bond to a reactive site of the immuno reactant, preferably a site, the loss of which has no or as little as possible effect on the desired immunological properties of the immuno reactant. Once again, as regards a fuller description of the bifunctional reagents, reference is made to the description of the process further below.
Enzyme-linked immunosorbents for use in assays (Elisa) for specific antibodies are known (A Voller et alia, proc. soc. exp. biol. med. 163, 402-5 (1980)). Immunoreactants bonded to a substrate by adsorption or ion exchange had been known, but the bond is weak.
Sportsman and Wilson (Anal. Chem. (1980), 52, 2013-2018) describe antibodies covalently bonded via their protein amine groups (by Schiff reaction followed by reduction with sodium borohydride) to aldehyde groups formed by periodate oxidation of glycidoxypropylsilane groups chemically linked to the silica atoms of silanised porous glass micro beads serving as carrier bodies. The porous glass beads having a diameter of 10 micron are silanised by immersion in a solution of glycidoxypropyltrimethoxysilane (GOPS). This results in the individual silane molecules attaching themselves chemically to individual silicon atoms of the silica of the glass. The organic glycidoxypropyl groups are converted by oxydation into aldehyde groups which in turn are reacted with the antibodies which are thereby immobilised. Immunosorbtive characteristics of the resulting carrier-bound immunosorbents are studied by observing the performance of chromatographic columns packed with the micro beads. For that purpose two antigens (human IgG and beef insulin) are first immunosorbed and then released by lowering the pH and with acetonitrile respectively. The immunosorbent is prepared specially using a different antibody for each antigen. Neither anti-insulin nor anti-human IgG antibodies are suitable for manufactured immunosorbents having a wide spectrum of uses. The method is of limited scope as regards chemical groups (amino) of an immunoreactant which can be used for the immobilising reaction as well as the geometry of the linkage and in particular the chemical and physical nature of the carrier body and its surface. It is not always desirable that the carrier body should have a silicious surface or have the physical properties of silica or glass (e.g. if metallic or magnetic properties would be useful). Also it is not always desired that the immobilised member of an antibody/hapten (antigen) pair in an immunosorption reaction should necessarily be the antibody.
The prior art silanisation of the glass particles will result in a "covering" of the glass surfaces which will normally be no more than monomolecular but may be even less. Such a "covering" will not as a rule avoid non-specific and easily reversible absorption of test substances, and this will interfere with desired forms of specific and strong immunosorption. Exposed siliceous adsorption sites and polar groups on glass surfaces tend to denature sensitive proteinaceous compounds. The porosity of the prior art microbeads, whilst desirable for some purposes, is a distinct disadvantage in the preferred field of application of the present invention. It influences the surface area unpredictably and in a manner which may vary from one bead to another. Such porosity has a molecular sieve effect whereby exclusion and molecular size-dependent retention effects are superimposed on and interfere with the desired selective and pure immunosorbtive effects. The smallness of the prior art beads makes them unsuitable for specific uses which the present invention contemplates.
The prior art carrier-bound immunosorbents each lack one or more of the following desirable qualities to a greater or lesser degree: stability, universal applicability, high capacity, ease of standardisation.